Expanding the Diversity of Plant Monoterpenoid Indole Alkaloids Employing Human Cytochrome P450 3A4

Human drug-metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R,20R)-epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.     

β-Diketonate versus β-Ketoiminate: The Importance of a Ferrocenyl Moiety in Improving the Anticancer Potency

Herein we present a library of fully characterized β-diketonate and β-ketoiminate compounds that are functionalized with a ferrocenyl moiety. Their cytotoxic potential has been determined by screening against human breast adenocarcinomas (MCF-7 and MDA-MB-231), human colorectal carcinoma p53 wild type (HCT116 p53^+/+) and normal human prostate (PNT2) cell lines. The ferrocenyl β-diketonate compounds are more than 18 times more cytotoxic than the ferrocenyl β-ketoiminate analogues. Against MCF-7, compounds functionalized at the meta position are up to nine times more cytotoxic than when functionalized at the para position. The ferrocenyl β-diketonate compounds have increased selectivity towards MCF-7 and MDA-MB-231, with several complexes having selectivity index (SI) values that are more than nine times (MCF-7) and more than six times (MDA-MB-231) that of carboplatin. The stability of these compounds in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) has been assessed by NMR spectroscopy and mass spectrometry studies, and the compounds show no oxidation of the iron center from Fe^II to Fe^III. Cytotoxicity screening was performed in both DMSO and DMF, with no significant differences observedin their potency.     

DPP-4 Cleaves α/β-Peptide Bonds: Substrate Specificity and Half-Lives

The incorporation of β-amino acids into a peptide sequence has gained particular attention as β- and α/β-peptides have shown remarkable proteolytic stability, even after a single homologation at the scissile bond. Several peptidases have been shown to cleave such bonds with high specificity but at a much slower rate compared to α-peptide bonds. In this study, a series of analogs of dipeptidyl peptidase-4 (DPP-4) substrate inhibitors were synthesized in order to investigate whether β-amino acid homologation at the scissile bond could be a valid approach to improving peptide stability towards DPP-4 degradation. DPP-4 cleaved the α/β-peptide bond after the N-terminal penultimate Pro with a broad specificity and retained full activity regardless of the β³-amino acid side chain and peptide length. Significantly improved half-lives were observed for β³Ile-containing peptides. Replacing the penultimate Pro with a conformationally constrained Pro mimetic led to proteolytic resistance. DPP-4 cleavage of α/β-peptide bonds with a broad promiscuity represents a new insight into the stability of peptide analogs containing β-amino acids as such analogs were thought to be stable towards enzymatic degradation.     

Metallocenyl 7-ACA Conjugates: Antibacterial Activity Studies and Atomic-Resolution X-ray Crystal Structure with CTX-M β-Lactamase

The conjugation of organometallic groups to current β-lactam antibiotics is a field of increasing study due to the ability of certain organometallic groups to enhance the antibiotic potency of these drugs. Herein, we report the antibacterial properties of two metallocenyl (ferrocenyl and ruthenocenyl) 7-aminocephalosporanic acid (7-ACA) antibiotic conjugates. Continuing a trend we found in our previous studies, the ruthenocenyl conjugate showed greater antibacterial activity than its ferrocenyl counterpart. Compared with the previously published 7-aminodesacetoxycephalosporanic acid (7-ADCA) conjugates, the 3-acetyloxymethyl group significantly improved the compounds’ activity. Furthermore, the Rc-7-ACA compound was more active against clinical Staphylococcus aureus isolates than the ampicillin reference. Noticeably, neither of the two new compounds showed an undesirable toxic effect in HeLa and L929 cells at the concentrations at which they displayed strong antibacterial effects. The antibacterial activity of the two metallocenyl 7-ACA derivatives was further confirmed by scanning electron microscopy (SEM). SEM micrographs showed that bacteria treated with metallocenyl 7-ACA derivatives feature cell wall damage and morphology changes. Using a CTX-M-14 β-lactamase competition assay based on nitrocefin hydrolysis, we showed that the Rc-7-ACA bound more favorably to CTX-M-14 than its ferrocenyl counterpart, again confirming the superiority of the ruthenocenyl moiety over the ferrocenyl one in interacting with proteins. We also report a 1.47 Å resolution crystal structure of Rc-7-ACA in complex with the CTX-M-14 E166A mutant, an enzyme sharing a similar active site configuration with penicillin-binding proteins, the molecular target of β-lactam antibiotics. These results strengthen the case for the antibacterial utility of the Rc and Fc groups.     

Recent Development of Photothermal Agents (PTAs) Based on Small Organic Molecular Dyes

Photothermal therapy (PTT) has attracted great attention due to its noninvasive and effective use against cancer. Various photothermal agents (PTAs) including organic and inorganic PTAs have been developed in the last decades. Organic PTAs based on small-molecule dyes exhibit great potential for future clinical applications considering their good biocompatibility and easy chemical modification or functionalization. In this review, we discuss the recent progress of organic PTAs based on small-molecule dyes for enhanced PTT. We summarize the strategies to improve the light penetration of PTAs, methods to enhance their photothermal conversion efficiency, how to optimize PTAs’ delivery into deep tumors, and how to resist photobleaching under repeated laser irradiation. We hope that this review can rouse the interest of researchers in the field of PTAs based on small-molecule dyes and help them to fabricate next-generation PTAs for noninvasive cancer therapy.     

Intersecting Xenobiology and Neometabolism To Bring Novel Chemistries to Life

The diversity of life relies on a handful of chemical elements (carbon, oxygen, hydrogen, nitrogen, sulfur and phosphorus) as part of essential building blocks; some other atoms are needed to a lesser extent, but most of the remaining elements are excluded from biology. This circumstance limits the scope of biochemical reactions in extant metabolism – yet it offers a phenomenal playground for synthetic biology. Xenobiology aims to bring novel bricks to life that could be exploited for (xeno)metabolite synthesis. In particular, the assembly of novel pathways engineered to handle nonbiological elements (neometabolism) will broaden chemical space beyond the reach of natural evolution. In this review, xeno-elements that could be blended into nature's biosynthetic portfolio are discussed together with their physicochemical properties and tools and strategies to incorporate them into biochemistry. We argue that current bioproduction methods can be revolutionized by bridging xenobiology and neometabolism for the synthesis of new-to-nature molecules, such as organohalides.     

Breaking a Couple: Disulfide Reducing Agents

Cysteine is present in a large number of natural and synthetic (bio)molecules. Although the thiol side chain of Cys can be in a free form, in most cases it forms a disulfide bond either with a second Cys (bridge) or with another thiol, as in the case of protecting groups. Efficient reduction of these disulfide bridges is a requirement for many applications of Cys-containing molecules in the fields of chemistry and biochemistry. Here we review reducing methods for disulfide bonds, taking into consideration the solubility of the substrates when selecting the appropriate reducing reagent.     

Iron-Based Nanozymes in Disease Diagnosis and Treatment

Iron-based nanozymes are currently one of the few clinical inorganic nanoparticles for disease diagnosis and treatment. Overcoming the shortcomings of natural enzymes, such as easy inactivation and low yield, combined with their special nanometer properties and magnetic functions, iron-based nanozymes have broad prospects in biomedicine. This minireview summarizes their preparation, biological activity, catalytic mechanism, and applications in diagnosis and treatment of diseases. Finally, challenges to their future development and the trends of iron-based nanozymes are discussed. The purpose of this minireview is to better understand and reasonably speculate on the rational design of iron-based nanozymes as an increasingly important new paradigm for diagnostics.     

Development of Core-Shell Nanoparticle Drug Delivery Systems Based on Biomimetic Mineralization

Among various drug-delivery systems, core-shell nanoparticles have many advantages. Inspired by nature, biomimetic synthesis has emerged as a new strategy for making core-shell nanoparticles in recent years. Biomimetic mineralization is the process by which living organisms produce minerals based on biomolecule templating that leads to the formation of hierarchically structured organic–inorganic materials. In this minireview, we mainly focus on the synthesis of core-shell nanoparticle drug-delivery systems by biomimetic mineralization. We review various biomimetic mineralization methods for fabricating core-shell nanoparticles including silica-based, calcium-based and other nanoparticles, and their applications in drug delivery. We also summarize strategies for drug loading in the biomolecule-mineralized core-shell NPs. Current challenges and future directions are also discussed.     

Assembly of a DNA Origami Chinese Knot by Only 15% of the Staple Strands

As a giant leap in DNA self-assembly, DNA origami has exhibited an unprecedented ability to construct nanostructures with arbitrary shapes and sizes. In typical DNA origami, hundreds of short DNA staple strands fold a long, single-stranded (ss) DNA scaffold cooperatively into designed nanostructures. However, large numbers of DNA strands are expensive and would hinder applications such as pharmaceutical investigations because of the complicated components. Therefore, one challenge is how to reduce the number of staple strands needed to construct DNA origami. For a DNA origami structure, the scale-free folding pattern of the scaffold strand is determined by staple strands at the branching vertexes. Simple duplex regions help to define the size-related features of the origami geometry. In this study, we hypothesized that a scaffold strand can be correctly folded into a designed topology by using only staple strands involved in branching vertexes. After assembly, any remaining, flexible, single-stranded regions of the scaffold could be converted into rigid duplexes by DNA polymerase to achieve the designed geometric structures. To demonstrate the concept, we used only 18 staple strands (covering 15 % of the scaffold strand) to assemble a porous DNA nanostructure, which was visualized by atomic force microscopy (AFM). This study helps understanding of the role of cooperativity in origami folding, and provides a cost-effective approach for small-scale prototyping DNA origami.